Eukaryotic cell motility is essential for many physiological processes such as embryonic development, tissue renewal, and the function of the immune system. Dictyostelium discoideum has proven to be an excellent model for the chemotactic migration of amoeboid cells such as leukocytes. Amoeboid migration is the result of the sequential repetition of pseudopod protrusions and retractions and is driven by the generation of traction forces. The strength and spatiotemporal organization of traction forces is determined by the coordinated interactions of actin-directed motors, F-actin regulation, actin crosslinking, motor protein-mediated contractility, and cell adhesions. However, precise knowledge of the biophysical coordination of these processes has been limited by the lack of quantitative information and analysis. We and others have observed that a considerable portion of the changes in cell shape occurring during amoeboid migration are due to periodic repetitive events, which enables the use of conditional statistics methods to analyze the network of biochemical processes involved in cell motility. The primary goal of this research is to determine in a statistically-robust, quantitative manner the biochemical basis for the spatiotemporal distribution of traction forces and the duration of each phase of the motility cycle by studying the role of candidate cytoskeletal and regulatory molecules with known or suspected involvement in the different stages of the motility cycle. We hypothesize that Myosin II is essential not only to the contractility phase of the motility cycle but also to the pseudopod protrusion phase. The generation of the traction forces depends not only on the contractile action of Myosin II, but also in its actin crosslinking effect. Based on preliminary results, we further hypothesize that the spatiotemporal distribution of traction forces and the average distance a cell travels per cycle depend on actin polymerization. To test the above hypotheses, we propose three Specific Aims. Specific Aim 1 is to apply our new 3D force cytometry method to measure the three components of the forces exerted by the cells on the substrate. The second and third aims are aimed at studying the role of candidate cytoskeletal and regulatory molecules with known or suspected involvement in the motility by undertaking systematic comparison of wild type cells and mutant strains with actin crosslinking or motor protein contractility defects (Specific Aim 2), and F-actin regulation defects (Specific Aim 3). Our method consists of simultaneously measuring the spatial and temporal changes in the distribution of fluorescently tagged signaling (or cytoskeletal) proteins and the 3D traction forces that mediate each stage of the cell motility cycle, while also recording the changes in cell shape. We will apply conditional statistics and Principal Component Analysis (PCA) to connect specific biochemical processes to each of the physical events in the motility cycle. Our studies will provide the necessary building blocks to begin constructing the complex network of biochemical processes controlling cell migration. PUBLIC HEALTH RELEVANCE: Motility of eukaryotic cells is essential for many physiological processes such as embryonic development, and tissue renewal, as well as for the function of the immune system. Incorrect regulation of motility plays an important part in many diseases (cancer, destructive inflammation, osteoporosis, mental retardation, etc.), and therefore, future therapeutic approaches will benefit from a precise quantitative understanding of the biophysical processes controlling cell motility. The aim of this study is to establish the mechanisms whereby each individual stage of the motility cycle is related to specific biochemical signaling events, and to elucidate the effects that the regulation of these signaling pathways has on cell motility, with the ultimate goal of developing a level of understanding of the biomechanical processes sufficient to predict and control cell motility.